Approximately one of every 247 (over 1.1 million people) people is legally blind in the United States, Worldwide it is estimated that 42,000,000 people are affected by blindness—either total or nearly so. Additional large numbers of people suffer from other severe retinal disorders.
Blindness in the developing world is often preventable. For example, a study of blindness in India reveals that 62% is caused by cataracts, 19% by refractive error, 5.8% by untreated glaucoma.
However, retinal disorders, including without limitation, diabetic retinopathy, retinitis pigmentosa (RP), wet and dry age-related macular degeneration (ARMD), inflammatory disease including macular edema, central vein occlusion, uveitis affecting the retina, and proliferative vitreoretinopathy are much more prevalent causes of blindness in the Western world.
Diabetic retinopathy is another common form of retinal disease. While diet, exercise, and drug therapy can do much to lessen the ocular effects of diabetes on the retina, there is no specific cure or prophylactic for diabetic retinopathy.
Similarly, glaucoma is a condition that is most commonly (though not exclusively) characterized by high intraocular pressure and which also involves degeneration of the retinal and optic nerve. While high intraocular pressure is susceptible to management with, for example, β adrenergic receptor antagonists such as timolol and α adrenergic receptor agonists such as brimonidine, the neural degeneration that accompanies glaucoma is neither reversible nor can it be definitively halted by lowering intraocular pressure alone.
In the developed world, by far the major retinal disease causing blindness in adults over 60 is age related macular degeneration (AMD), and with the segment of the population within this age range steadily increasing in the United States, the number of cases are likely to increase by the same rate without an effective treatment for the condition.
AMD progressively decreases the function of specific neural and epithelial layers of the retinal macula. The clinical presentation of the condition includes the presence of drusen, hyperplasia of the retinal pigmented epithelium (RPE), geographic atrophy, and choroidal neovascularization (CNV). Atrophic AMD is characterized by outer retinal and RPE atrophy and subadjacent choriocapillaris degeneration, and accounts for about 25% of cases with severe central visual loss.
Exudative (or “wet”) AMD is characterized by CNV growth under the RPE and retina, and subsequent hemorrhage, exudive retinal detachment, diciform scarring, and retinal atrophy. Pigment epithelial detachment can also occur. Exudative AMD accounts for about 75% of AMD cases with severe central vision loss.
Currently most treatment for this disease involves therapies that are most helpful to patients who are suffering from relatively advanced symptoms of the disease. These therapies include laser photocoagulation, photodynamic therapy and surgery in cases where CNV is involved. However, there is no currently effective therapy for the early stages of the disease.
It is known that inflammation, particularly chronicchromic inflammation, plays a large part in the development of AMD. Drusen, the presence of which is one of the hallmarks of AMD, comprise protein and cellular components including immunoglobulin and components of the complement pathways that are involved in immune complex deposition, molecules involved in acute response to inflammation such as α1-antitrypsin and amyloid P component; major histocompatibility complex class II antigens. Additionally, drusen include RPE fragments, melanin and lipofuscin. Some researchers have suggested that the presence of vitronectin, apolipoprotein E and other drusen-associated molecules indicates that the RPE cells are subject to a chronic, sub-lethal complement attack. Such an attack may result in the elimination of surface-associated membrane attack complexes (such as by shedding or endocytosis of cell membrane) and the formation of extracellular deposits of immune complexes and complement components, activated macrophages and other inflammatory cells secrete enzymes that can damage cells and degrade the Bruch membrane (the inner layer of the choroid in contact with the RPE). By releasing cytokines, inflammatory cells may encourage CNV growth in the sub-RPE space.
Interestingly, complement activation and associated inflammatory events occur in other diseases that exhibit cellular degeneration and accumulation of abnormal tissue deposits, such as arthrosclerosis and Alzheimer disease. Indeed, the Alzheimer β-amyloid peptide is found together with activated complement components in a sub structural vesicular component with drusen.
Intravitreal administration of corticosteroid appears to reduce the incidence of CNS infiltration in primates. This may be due to the known anti-inflammatory activity of steroids, which may alter inflammatory cell activity in the choroid. However, chronic use of steroids can have serious side effects, including glucose intolerance, diabetes and weight gain.
Initiation of an inflammatory response involves the detection of an injury, other insult, or infection by members of the host immune surveillance system, comprising immune cells that are involved in trafficking around the body. Immune cell trafficking involves circulation, homing and adhesion, extravasation (entry of the leukocyte through the endothelial barrier), and movement of particular populations of leukocytes between the blood vessels, lymph and lymphatic organs and the tissues.
Trafficking is regulated by a complex interaction of cellular adhesion molecules ((such as integrins and selectins) and of a family of cytokines, termed chemokines, and their receptors.
Chemokines comprise a large family of chemoattractant molecules that function in part to guide phagocytotic leukocytes of the immune system to injured or infected tissue. Two groups of chemoattractants have been identified to date; the first group comprises “classical” chemoattractants including bacterially-derived N-formyl peptides, complement fragment peptides C5a and C3a, and lipids such as leukotriene B4 and platelet-activating factor.
The second, more recently characterized group of chemoattractants comprises a superfamily of chemotactic cytokines having molecular weights of from about 8 to about 17 KDa. These chemokines are secreted proteins that function in leukocyte trafficking, recruiting, and recirculation. They have also been discovered to play a critical role in many pathophysiological processes such as allergic responses, infectious and autoimmune diseases, angiogenesis, inflammation, tumor growth, and hematopoietic development. Approximately 80 percent of these proteins have from 66 to 78 amino acids in their mature form comprising a core region of relative homogeneity. The remaining chemokines are of larger molecular weight, with additional amino acids occurring upstream of the protein “core”, or as part of an extended C-terminal segment.
All chemokines signal through the chemokine subfamily of seven transmembrane domain G-protein coupled receptors (GPCRs). GPCRs constitute the single largest family of signal detectors at the cell surface. Activation of GPCRs by selective or specific ligands triggers signal propagation via the G proteins, which subsequently regulate the activities of downstream effector molecules within the target cell. G proteins are so named because they can bind to and are activated by guanidine triphosphate (GTP).
The fidelity of GPCR-mediated signal transduction is maintained at several levels. Firstly, the ligand-receptor interaction is highly selective where discrimination of ligand stereoisomers is commonly observed. Secondly, each GPCR can generally only interact with a small subset of G proteins, which in turn regulate a limited number of effectors. The G proteins are classified into four subfamilies termed Gs, Gi, Gq and G12, according to their sequence homologies.
The intact G holoproteins are heterotrimeric polypeptides. The guanidine diphosphate (GDP)-bound form of the heterotrimeric G protein is inactive, while the GTP-bound form is active. Upon ligand binding to the GPCR, the receptor undergoes a change in conformation that results in the recruitment of the inactive heterotrimeric G protein to the ligand-bound GPCR. Once bound to the receptor, the α subunit of the G protein expels the bound GDP, replaces the GDP with GTP and, so activated, the α subunit of the G protein now dissociates from the tightly associated Gβ and Gγ subunit (or “βγ”) dimer. The βγ dimer is then free to interact with and regulate various effectors. Similarly, the activated α subunit can then, for example, bind to and stimulate adenylyl cyclase, that in turn regulates the catalytic production of cAMP. Alternatively, if the G protein is a Gq trimer, the activated α subunit can bind to and regulate PLC.
The primary structures of all the Gq family α subunits share high percentages of identity with each other and they also share common functional properties. They can regulate the activity of phospholipase C isoforms (PLC) through selective activation by GPCRs. This leads to an increase in the intracellular level of inositol phosphates (IP).
The GPCRs are members of the class of receptors known as “serpentine” receptors. Helical domains of these structurally related receptors cross the plasma membrane seven times and possess an extracellular amino terminus and intracellular carboxyl terminus. G protein-coupled receptors are estimated to occur in more than 1000 variations in mammals and regulate some activity in nearly every human cell. Members of the G protein-coupled receptor superfamily include, without limitation, the alpha adrenergic, beta-adrenergic, dopamine, muscarinic, acetylcholine, nicotinic acetylcholine, rhodopsin, opioid, somatostatin, and serotonin receptors.
There are currently at least seventeen known chemokine receptors, and many of these receptors exhibit promiscuous binding properties, whereby several different chemokines can signal through the same receptor. The chemokine receptors are approximately 350 amino acids in length and can be aligned with each other only if gaps are introduced into the primary “universal” sequence. The N terminus is acidic and extracellular can be sulfated and contain N-linked glycosylation sites. The C terminus is intracellular and comprises serine and threonine residues capable of being phosphorylated for receptor regulation. The seven transmembrane domains are linked by three intracellular and three extracellular loops of hydrophilic residues. The highly conserved cysteines in the 1st and 2nd extracellular loops are joined in a disulfide bond. The G proteins couple to the receptors by way of the C-terminus and perhaps the third intracellular loop.
The chemokine receptor ligands are divided into subfamilies based on conserved amino acid sequence motifs. Most chemokine family members have at least four conserved cysteine residues that form two intramolecular disulfide bonds. The subfamilies are defined by the position of the first two cysteine residues, Thus:
a) The alpha (α) subfamily, also called the CXC chemokines, have one amino acid (“X”, designating that any amino acid may occupy this position) separating the first two cysteine residues. This group can be further subdivided based on the presence or absence of a Glu-Leu-Arg (ELR) amino acid motif immediately preceding the first cysteine residue. There are currently five CXC-specific receptors and they are designated CXCR1 to CXCR5. The ELR chemokines bind to CXCR1 and/or CXCR2 and generally act as neutrophil chemoattractants and activators. At present, 14 different human genes encoding CXC chemokines have been reported in the scientific literature with some additional diversity contributed by alternative splicing.
b) In the beta (β) subfamily, also called the CC chemokines, the first two cysteines are adjacent to one another with no intervening amino acid. There are currently 24 distinct human β subfamily members. The receptors for this group are designated CCR1 to CCR11. Target cells for different CC family members include most types of leukocytes.
c) There are two known proteins with chemokine homology that fall outside of the α and β subfamilies. Lymphotactin is the lone member of the gamma (γ) class (C chemokine) which has lost the first and third cysteines. The lymphotactin receptor is designated XCR1. Fractalkine, the only currently known member of the delta (δ) class (CXC chemokine), has three intervening amino acids between the first two cysteine residues. This molecule is unique among chemokines in that it is a transmembrane protein with the N-terminal chemokine domain fused to a long mucin-like stalk. The fractalkine receptor is known as CXCR1.
A variety of approaches have been used to identify chemokines. The earliest discoveries of chemokines were made as a result of their biological activity or through studies that sought to identify proteins that are upregulated following cell activation or differentially expressed in selected cell types. Most of the recently reported chemokines, however, were identified through bioinformatics. EST (Expressed Sequence Tags) databases contain the sequences of a large number of cDNA fragments from a variety of tissues and organisms. Translation of ESTs can provide partial amino acid sequences of the proteome. Because the chemokines are comparatively small and contain signature amino acid motifs, many novel family members have been identified through searches of EST databases.
The stromal cell-derived factors SDF-1α and SDF-1β are CXC chemokines encoded by alternatively spliced mRNAs. The mature α and β forms differ only in that the β form has four additional amino acids at its C-terminus. These proteins are highly conserved between species. Most functional studies have been performed with SDF-1α and suggest a variety of roles for this molecule. It is necessary for normal development of B cells and brain. It is a potent chemoattractant for CD34 bone marrow progenitor cells and dendritic cells. It also appears to play a role in trafficking and adhesion of lymphocytes and megakaryocytes.
An additional alternatively-spliced product from rat, designated SDF-1γ, was reported recently. The SDF-1γ mRNA is similar to the SDF-1β message but with an additional exon inserted near the C-terminal end of the coding region. The four amino acids of SDF-1β that are normally appended to the C-terminus of SDF-1α are replaced in SDF-1γ by a 30 amino acid segment containing 17 positively charged residues. The SDF-1β and 1γβ transcripts display different patterns of expression in a number of tissues. They are also reciprocally expressed in developing rat brain. SDF-1β is expressed in embryonic and neonatal brain, whereas SDF-1γ is expressed in adult brain. The function of this variant is still currently unknown.
A unique chemotactic activity was recently reported for SDF-1 in which subpopulations of T cells were attracted by SDF-1α concentrations of 100 ng/mL but repelled by concentrations of 1 μg/mL. The higher concentration that elicited repulsion is comparable to the concentration of SDF-1 that occurs in the bone marrow. Inhibitor studies reveal that migration in both directions requires the CXCR4 receptor, G-proteins, and phosphatidylinositol 3-kinase. However, tyrosine kinase inhibitors block chemoattraction and have no affect on chemorepulsion, whereas a cAMP agonist inhibits chemorepulsion but does not affect chemoattraction.
CD34 is a cell-surface marker that correlates in humans with bone marrow progenitors having a high proliferative response to hematopoietic cytokines. CD34+ hematopoietic progenitor cells (HPC) have been observed to migrate both in vitro and in vivo toward a gradient of SDF-1 produced by structural bone marrow cells called stromal cells. In the in vivo experiments, the SDF-1 was administered to the spleen. See Aiuti et al., 185 J. Exp. Med. 111 (Jan. 6, 1997); this and all other references cited in this patent application are hereby incorporated by reference herein in their entirety.
Myeloid and erythroid cells, as well as B- and T-lymphoid cells, have been found in cultures of cells having the CD34+ phenotype. The concentrations of SDF-1 that stimulate a chemotactic response also cause a transient elevation of Ca++ in CD34+ cells. CD34+ cells have been demonstrated to be able to reconstitute blood cells in lethally irradiated baboons and in humans. Pertussis toxin completely inhibited the SDF-1 induced chemotaxis, indicating that the chemokine receptor present in CD34+ cells processes SDF-1 signals through Gi. SDF-1 attracts CFU-GM (granulocyte/macrophage colony forming units), CFU-MIX (colony forming units of mixed lineage and BFU-E (erythroid burst-forming units) progenitor cells from human bone marrow (BM), umbilical cord blood (CB) and mobilized peripheral blood (PB). There is a high degree of conservation between human and mouse SDF-1 (a single amino acid difference), and mouse SDF-1 causes transendothelial chemotaxis on both human and mouse HPC.
Interestingly, HPCs have been identified as being capable of differentiating into liver cells. When clinical liver or bone marrow transplantation occurs, occasionally BM-derived hepatocytes have been found. While the number of HPCs that engraft irradiated liver and develop into hepatocyte-like, albumin-producing cells is extremely low, when the liver is injured or challenged by viral inflammation, the number of such cells increases in response to the stress. In mice there is a very large amplification of HPCs that have at least some of the hallmarks of hepatic morphology and function.
SDF-1, which is also known as CXCL12, is widely expressed in various tissues during both development an adulthood, and these tissues include the liver. Stress, caused by injury, infection or insult, can facilitate tissue-specific differentiation and induce secretion of signaling mediators that increase migration and guide transplanted pluripotent stem cells to the injured tissue. Also, there is increased expression of SDF-1 in the liver after the entire body is irradiated. In humans, those patients injected with HCV, expression of SDF-1 is extended to bile ductile tissue, canal of Hering, and oval cells.
This stress-related increase in SDF-1 expression appears to be associated with some factors including matrix metalloproteases MMP-9 and MMP-2. These proteases are activated following CCl4-mediated liver injury, and this phenomenon in turn triggers mobilization of human progenitor cells from the marrow into general circulation. At the same time the levels of CXCR4 receptor expression increase.
Guerciolini et al., U.S. Patent Application Publication 2006/0019917 discloses siRNA interference of a stromal cell derived factor-1 isoform.
All publications cited in this application are hereby incorporated by reference herein in their entirety, regardless whether an express incorporation by reference is made along with a citation of the reference or not.